Molecular diversity of phospholipase D in angiosperms

BACKGROUND: The phospholipase D (PLD) family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology. RESULTS: Taking advantage of sequence data available in public databases, we attempted to provide a prerequisite for such an approach. We made a complete inventory of the Arabidopsis thaliana PLD family, which was found to comprise 12 distinct genes. The current nomenclature of Arabidopsis PLDs was refined and expanded to include five newly described genes. To assess the degree of plant PLD diversity beyond Arabidopsis we explored data from rice (including the genome draft by Monsanto) as well as cDNA and EST sequences from several other plants. Our analysis revealed two major PLD subfamilies in plants. The first, designated C2-PLD, is characterised by presence of the C2 domain and comprises previously known plant PLDs as well as new isoforms with possibly unusual features-catalytically inactive or independent on Ca(2+). The second subfamily (denoted PXPH-PLD) is novel in plants but is related to animal and fungal enzymes possessing the PX and PH domains. CONCLUSIONS: The evolutionary dynamics, and inter-specific diversity, of plant PLDs inferred from our phylogenetic analysis, call for more plant species to be employed in PLD research. This will enable us to obtain generally valid conclusions

In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins

Calcium (Ca+2) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a ∼25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling

Calcium in health and disease.

Evolution has exploited the chemical properties of Ca(2+), which facilitate its reversible binding to the sites of irregular geometry offered by biological macromolecules, to select it as a carrier of cellular signals. A number of proteins bind Ca(2+) to specific sites: those intrinsic to membranes play the most important role in the spatial and temporal regulation of the concentration and movements of Ca(2+) inside cells. Those which are soluble, or organized in non-membranous structures, also decode the Ca(2+) message to be then transmitted to the targets of its regulation. Since Ca(2+) controls the most important processes in the life of cells, it must be very carefully controlled within the cytoplasm, where most of the targets of its signaling function reside. Membrane channels (in the plasma membrane and in the organelles) mediate the entrance of Ca(2+) into the cytoplasm, ATPases, exchangers, and the mitochondrial Ca(2+) uptake system remove Ca(2+) from it. The concentration of Ca(2+) in the external spaces, which is controlled essentially by its dynamic exchanges in the bone system, is much higher than inside cells, and can, under conditions of pathology, generate a situation of dangerous internal Ca(2+) overload. When massive and persistent, the Ca(2+) overload culminates in the death of the cell. Subtle conditions of cellular Ca(2+) dyshomeostasis that affect individual systems that control Ca(2+), generate cell disease phenotypes that are particularly severe in tissues in which the signaling function of Ca(2+) has special importance, e.g., the nervous system